Accurate and precise determination of viable sperm concentration is an important issue for the Artificial Insemination (AI) industry since it provides assurance to the studs and customers that the insemination doses contain the sperm numbers indicated. This is especially important in relation to exportation of semen from domesticated animal species (Foote, 1972; Fenton et al., 1990; Woelders 1990; Evenson et al., 1993; Donoghue et al., 1996).
It is known in the art to use counting chambers such as Makler™ (Sefi Medical, Haifa, Israel) or hemacytometers for routine sperm counts. However, multiple measurements are required to achieve an acceptable precision and accuracy. This procedure is time consuming and makes the counting chambers slow in use. Single hemacytometer counts are not highly accurate because of inherent errors in the technique, such as obtaining a subsample representative of the semen sample, etc, and furthermore, the process is highly dependant upon operator skills.
A technique widely used by the artificial insemination industry to determine sperm cell concentration is based on spectrophotometric measurement of turbidity (Woelders 1990; Evenson et al., 1993). However, this method requires a calibration of the spectrophotometer which is repeated at regular intervals since the performance of the instrument will vary over time, whereby this method is only as accurate as the calibration method. The calibration is most often based on one of the counting chambers mentioned above, whereby the spectrophotometer method is only as accurate as the counting chamber methods mentioned above.
A further disadvantage of the spectrophotometer method is that debris, such as gel-particles in boar semen, increases the semen turbidity whereby inaccuracies are introduced in the method since distinguishing debris from semen is very difficult (Woelders 1990) and the method is used only extensively because of the rapidity of the method and the fact that no other method works in a satisfactory manner for these species.
Semen from species without large gel-particles in the semen may be analyzed on electronic counters wherein particles having a specific particle size are counted (Parks et al., 1985). However, electronic counters are not capable of distinguishing semen cells from debris having a particle size corresponding to the particle size of the semen cells. Since semen from most species contains a high number of particles having a size corresponding to the size of spermatozoa (i.e. cytoplasmic droplets) the resulting counts for individual ejaculates can be highly inaccurate (Parks et al., 1985).
The evaluation of semen quality in artificial insemination stations as well as in laboratories for human semen and the evaluation of semen quality for semen from laboratory animals is normally based on an evaluation of sperm motility using a phase contrast microscope with a heated stage (Woelders, 1990). Although this procedure is valuable to ensure that semen of very poor quality is excluded from artificial insemination, the method does not have a high precision or accuracy. Furthermore, the method is highly dependent on operator skills and large variations are observed in the results obtained by different operators.
It is also known in the art to use flow cytometers for a characterisation of the sperm (Szollosi et al., 1986; Takacs et al., 1987; Evenson et al., 1993). In the methods disclosed, the sperm has been killed and stained with propidium iodide, PI, to measure sperm concentration. Thereby, only sperm concentration is found. It is a disadvantage of the method that no indication of the amount of live sperm cells is provided.
In another method, only the dead sperm cells are stained by propidium iodide, whereafter light scattered from the unstained particles (including live sperm) is detected. This method has been used by Takizawa et al. (1994, 1995 and 1998) as well as by Yamamoto et al. (1996). In this method, there is no discrimination between live spermatozoa and debris in the semen. Thereby, gel-particles, cytoplasmic droplets, debris and bacteria are likely to be included in the sperm count according to this method, thereby inflating the value of both the measured concentration and detected viability.
It is further known in the art to stain sperm cells using a fluorescent dye called SYBR-14 and propidium iodide, PI, (LIVE/DEAD® Sperm Viability Kit, Molecular Probes, Oregon, USA) and by means of flow cytometric analyses to asses the viability of the sperm (Garner et al., 1994; Garner et al., 1995; Donoghue et al., 1995; Garner et al., 1996a; Garner et al., 1996b; Garner et al., 1997a; Garner et al., 1997b Garner et al., 1997c; Maxwell et al., 1997; Maxwell and Johnson, 1997; Penfold et al., 1997; Songsasen et al., 1997; Thomas et al., 1997; Vetter et al., 1998; Thomas et al., 1998; Chalah and Brillard, 1998).
However, it is a disadvantage of this method that the SYBR-14 is slightly toxic to spermatozoa whereby the amount of live spermatozoa in a sample will decrease over even a short time. Since a staining time of 10 to 15 minutes has been used routinely in combination with 100 nM of SYBR-14, results are likely to be inaccurate due to the decrease in live spermatozoa over time (Christensen and Stenvang, unpublished data). It is a further disadvantage of the method that the incubation of samples for staining is made at 36–37° C., which is highly inconvenient in routine work in laboratories or artificial insemination stations assessing semen quality.
In U.S. Pat. No. 4,559,309 a process for characterization of sperm motility and viability is disclosed. A sperm sample is stained with Rhodamine 123 and ethidium bromide and the fluorescence emissions of the sperm are measured by means of a flow cytometer. By measuring fluorescence emissions at green and at red wavelengths a measure correlated with sperm motility (green counts) and dead or putative dead sperm cells (red counts), respectively, is obtained. Staining of a second sample with acridine orange provides a measure of the percentage of single and double stranded DNA in the sperm cells thereby indicating mature and immature sperm and somatic cells. This process does not provide an absolute measure of the semen concentration. Furthermore, two different staining procedures are necessary to obtain a measure of sperm motility and dead or dying sperm ells and a measure of the types of sperm cells in the sample.
Furthermore, staining of mitochondria with Rhodamine 123 and DNA with PI implies that two different cellular compartments are targeted. ‘Detached heads’ are observed commonly in semen from normal bulls (affecting 5.3±0.4% of the sperm; Barth & Oko, 1989) and the incidence of this defect can be much higher for individual bulls. With this condition, the connected midpiece and tail will be motile and stain with R123. The detached head will stain with PI and consequently the percentage of viable or dying sperm cells in the sperm population will be estimated inaccurately.
It is further known in the art to control the sample injection to electronic counters or flow cytometers so as to obtain a measure of the concentration. However, in these methods the control of the sample injections is affected by differences between instruments, and calibration is needed at regular intervals. One example of such an instrument is Partec Sperm Cell Counter™ (Partec GmbH, Munster, Germany) where performance is unacceptable for assessment of sperm concentration in bovine semen (Dumont et al., 1996).
Further, it is known to use a standardised bead solution for absolute counting of the sperm concentration; the performance of this method was, however, found unacceptable for assessment of sperm concentration (Dumont et al., 1996).
It is common to all of the above-mentioned methods that only the ratio of live sperm cells or the concentration of the dead or killed sperm cells is determined. To obtain a determination of the ratio of live sperm cells and a determination of the concentration of the semen an evaluation of the semen sample using at least two different methods is needed.
Furthermore, a long-standing goal of the cattle artificial insemination (AI) industry is to obtain a simple, precise and accurate method for examination of bull semen that correlates highly with fertility after AI. Routine assessment of semen volume, sperm concentration, motility and wave motion in an AI stud are valuable in order to discard semen of poor quality, but appear of little value to predict the fertility of individual males (Christensen et al. 1999). In recent years, computer aided sperm analysis (CASA; Budworth et al. 1988) have provided an objective assessment of sperm motility. Disadvantages of this technique such as poor precision, bias due to program settings and sperm migration during analysis (Christensen and Stryhn, 1997) limits the potential use.